Translational Role of Rodent Models to Study Ventilator-Induced Lung Injury

Mechanical ventilation is an important part of medical care in intensive care units and operating rooms to support respiration. While it is a critical component of medical care, it is well known that mechanical ventilation itself can be injurious to the lungs. Despite a large number of clinical and preclinical studies that have been done so far, there still exists a gap of knowledge regarding how to ventilate patients mechanically without increasing lung injury. Here, we will review what we have learned so far from preclinical and clinical studies and consider how to use preclinical models of ventilation-induced lung injury that better recapitulate the clinical scenarios.


Introduction
Mechanical ventilation (MV) is a critical component of clinical care. MV is frequently utilized in intensive care units (ICUs) for patients with respiratory insufficiency. Acute respiratory distress syndrome (ARDS) is a severe form of lung injury and seen in approximately 10% of patients in ICUs [1]. 35% of all patients in ICUs worldwide receive MV even if they do not have ARDS [2]. MV is also used as a part of general anesthesia for surgical procedures in patients with normal lung function. 230 million patients per year require MV for major surgery [3]. While MV is supposedly administered for medical benefit to patients, it was recognized soon after its use that it could also cause structural lung damage (ventilator-induced lung injury; VILI) [4]. Thus, mitigating the chance of developing VILI is critical.
While clinical outcome studies are a must to understand existing issues, direct inspection of the lungs in patients undergoing MV to determine the nature and the extent of lung injury is almost impossible and not practical. The current diagnosis of lung injury is based on the clinical criteria such as the ratio of arterial oxygen partial pressure (PaO 2 in mmHg) to fractional inspired oxygen (FiO 2 expressed as a fraction) and imaging studies [5]. As a result, there are significant limitations in understanding the underlying mechanism of VILI development from clinical data. In addition, patient medical history and clinical scenarios for MV requirement are extremely diverse. In contrast, preclinical VILI models are advantageous in dissecting the mechanism of VILI development; Experimental conditions can be strictly controlled, and tissue samples are easily obtainable. A host of species has been used for preclinical studies. However, the lung anatomy, the mechanics and the immune system are not exactly the same among different species and therefore, no particular species/strategy combination developed to date can be described as a gold standard [6]. Among them, rodents are most popularly used. Thus, here we will review the role of preclinical rodent models in MV-induced lung injury research, particularly using mice.

Differences between Mouse and Human Lungs Anatomical differences
The mouse lung is quite different from human lung from a structural standpoint [7]. The total lung capacity (TLC) of the mouse is about 1 mL. For human adult, it is about 5,000-6,000 mL. Mouse lung has four right lobes and one left lobe, while human lung has three right lobes and two left lobes. Mouse lung has fewer respiratory bronchioles and airway generations (13)(14)(15)(16)(17) generations) compared to human lung (17-21 generations). The parenchyma of the mouse lung occupies a bigger fraction of the total lung (18%) than that of the human (12%). The alveoli of the mouse lung are smaller (80 μm mean linear intercept (MFI)) than those of the human (210 μm). The blood-air barrier thickness in the mouse is smaller (0.32 μm) compared to that of the human (0.62 μm). The anatomical differences between mouse and human lungs are summarized in Table 1.
important. Major professional phagocytes include neutrophils, monocytes, macrophages and dendritic cells. Intravascular macrophages directly face the circulation for microbial ingestion. In many species, which include rodents and human, intravascular macrophages are restricted to the spleen and liver [11]. In livestock species such as sheep and pigs, however, the lungs contain pulmonary intravascular macrophages, a resident population of mature macrophages that adhere to endothelial cells in pulmonary capillaries in addition to alveolar macrophages that usually exist in most species. How these anatomical differences in immune cell profiles among species affect microbial protection is not known, but knowing these differences and potentially delineating their contribution, is an important consideration when we test two-hit models where the first hit is triggered by microbial infection.
From genomic standpoint, mouse genome is quite similar to human. The genome sequence project of mouse on the C57/BL6 background indicates that its genome has actually 99% similarity to the human genome [12]. Although there was a study raising concerns that genomic responses in mouse models poorly mimic human inflammatory diseases [13], a follow-up study using the same database suggested that genomic responses in mouse models greatly mimic human inflammatory diseases [14]. However, it is certain that there are some immunological differences between mice and humans. For example, differences have been found in Toll-like receptors (TLRs), which are one of the pattern recognition receptors for microbial products and endogenous danger signals. Specifically, TLR4 from humans and mice has been found to recognize different lipopolysaccharide (LPS) structures [15]. In addition, intra-species differences in the immunological responses are important and should be taken into consideration to human-mice differences. C57/BL6 is one of the major strains that have been used in immunology research, but it is also important to know that are differences in the immunological responses among different mouse strains when testing VILI in different strains. For instance, and as described in more details below, A/J strain is more sensitive to VILI compared to C57/BL6. As mentioned above, TLR4 pathway is described to be important in VILI, and the intra-species difference in TLR4 signaling pathway may need to be given another consideration regarding immunological responses [16].

Ventilator-induced lung injury in ICU patients
From early days of MV use, structural damage to the lungs by MV was well recognized [17]. This includes pneumothorax, pneumomediastinum, and subcutaneous emphysema [18]. Although the term VILI was only introduced in 1993 [19], various lines of preclinical investigation were done which proposed that excessive pressure ("barotrauma") [20], excessive volume ("volutrauma") [21] and the cyclic opening-closing of the lung units subjected to atelectasis ("atelectrauma") [22] and biotrauma were considered four main causes for VILI as described in the following section. Subsequently, a number of clinical studies have been designed to test these concepts. Traditional MV regimen for Acute Respiratory Distress Syndrome (ARDS) was to use TV of 10-15 mL/kg [23]. The ARDS Network trial (ARMA trial) examined the outcome of patients with ARDS receiving lower and higher TVs to determine if higher TV would cause stretch-induced lung injury. The study demonstrated that the lower TV arm (6 mL/kg, mean plateau pressure 25 cmH 2 O) was associated with less mortality compared to the higher TV arm (12 mL/kg, mean plateau pressure 33 cm H 2 O) (mortality 31.0% vs. 39.8%) [23], supporting the idea that the ventilatory method could influence the outcome of patients. This result was in line with the concept of volutrauma and barotrauma. High positive end-expiratory pressure (PEEP) may also attenuate atelectrauma. Furthermore, IL-6 levels were higher in patients with mortality, supporting the concept of biotrauma in this patient cohort. Following this study, "lung protective" MV strategy using TV of approximately 6 mL/kg has been a common approach in patients with ARDS. Since the same TV can be attained under different pressures to the lungs, the role of pressure load posed to the lungs by MV in outcomes was also studied. Driving pressure is defined as a plateau pressure minus positive end-expiratory pressure (PEEP) (Pdriving = Pplat -PEEP) ( Figure 1). Plateau pressure is measured at the end of an inspiratory pause during volume-controlled ventilation and at the end of inspiration during pressure-controlled ventilation [24]. An inspiratory pause greater than or equal to 3 seconds predicts plateau pressure with the best accuracy [25]. In contrast, an inspiratory pause of 0.5 second overestimates plateau pressure by 11% in ARDS patients [25]. At a fixed tidal volume, changes in driving pressure that occur as PEEP increases or decreases, reflect changes in respiratory system compliance. At the PEEP associated with the lowest driving pressure, respiratory system compliance is the highest. So PEEP can be titrated to attain this goal. Because driving pressure consists of the pressure distributed to the lungs themselves (transpulmonary pressure) and the pressure applied to the chest wall, it is not fully representative of the stress forced to the lungs. Nonetheless, in the study by Amato, et al., higher driving pressure was associated with an increased mortality in patients with ARDS, while PEEP or plateau pressure did not show any association with the mortality [26]. Relative risk of in-hospital death was 1.0 at the driving pressure of around 15 cmH 2 O. The role of driving pressure in outcomes was also examined by Fuller, et al. in patients who required MV but did not have ARDS at the initiation of MV. The mortality and ARDS development were used as outcome measures. Non-survivors were significantly associated with higher driving pressures (15.9 vs. 14.9 cmH 2 O) and higher plateau pressures (21.4 vs. 20.4 cmH 2 O) than survivors [27]. In theory, a transpulmonary pressure should be taken into consideration rather than the driving pressure alone due to the reason described above. Nevertheless, these studies demonstrated that the driving pressure was an important parameter to consider for MV. On average, the ratio of lung elastance to total respiratory system (lung and chest wall) is 0.7. If the driving pressure is 20 cmH 2 O, transpulmonary pressure is 14 cmH 2 O. But the ratio can range from 0.2 to 0.8 [28]. The transpulmonary pressure is the same 14 cmH 2 O when the driving pressure is 28 cmH 2 O and the ratio is 0.5. Transpulmonary pressure is the pressure difference between the alveoli and the esophagus, requiring an esophageal pressure probe. The use of esophageal probe was shown to significantly improve oxygenation and compliance during MV in ARDS patients [29], though this measurement is not a routine in the clinical practice. Overall, a number of clinical studies in ICU patients has indicated that MV strategies could have an impact on patient outcomes.

Ventilator-induced lung injury in the operating room settings
MV is also utilized for general anesthesia during surgical procedures. In the operating room setting, patients who may have normal lung function prior to surgery receive MV for a short period [30]. Postoperative respiratory complications represent the second most common perioperative complication after wound infection, with an estimated incidence of 3.0-10.0% [31][32][33][34]. Because the majority of patients requiring MV in the ICU settings has pre-existing lung injury, the deterioration of respiratory status following MV may not be necessarily attributed to MV. In this sense, surgical patients may be more homogeneous from pre-MV lung wellbeing and easier in order to understand VILI.
A number of studies have examined the association between MV settings and postoperative complications. Futier, et al. performed the Intraoperative Protective Ventilation (IMPROVE) prospective trial to determine whether protective ventilation could improve outcomes after elective abdominal surgery [35]. In the study, patients aged 40 years and older received volume-controlled MV either with TV of 10-12 mL/kg, no PEEP, no recruitment maneuvers or with TV of 6-8 mL/kg, PEEP of 6-8 cmH 2 O and recruitment maneuvers every 30 minutes (lung protective ventilation). The primary outcome was a composite of major pulmonary (pneumonia, postoperative need for invasive or noninvasive ventilation) and extrapulmonary (sepsis, death) complications by postoperative day 7. Lung protective ventilation was associated with significantly less postoperative composite complications. Severgnini, et al. also prospectively examined the role of lung protective ventilation in open abdominal surgery [36]. In this study, patients aged 18 years and older received volume-controlled ventilation either with TV of 9 mL/kg, no PEEP or TV of 7 mL/kg, PEEP of 10 cmH 2 O and recruitment maneuvers (lung protective). Lung protective ventilation was associated with higher modified Clinical Pulmonary Infection Score (mCPIS) and better oxygenation on postoperative day 1 and 3. These studies paired high PEEP with low TV. To understand the role of PEEP in low TV ventilation, the Protective Ventilation using High versus Low PEEP (PROVHILO) trial was performed in open abdominal surgery [37]. Patients aged 18 years and older were enrolled for ventilation with TV of 8 mL/kg, PEEP of 12 cmH 2 O and recruitment maneuver or the same TV and PEEP < 2 cmH 2 O. The primary outcome was a composite of postoperative pulmonary complications (hypoxemia, bronchospasm, suspected pulmonary infection, pulmonary infiltrate, aspiration pneumonitis, ARDS, pleural effusion, pulmonary edema, pneumothorax) within the first 5 days after surgery. No difference in the incidence of postoperative pulmonary complications was observed between the two groups. However, this study did not clarify if PEEP level between 2 and 12 cmH 2 O had any role or not. In addition, it is important to point out that PROVHILO trial did not include laparoscopic procedures or morbidly obese patients, different from IMPROVE trial. Realizing these differences, these studies demonstrated the importance of low TV ventilation in patients undergoing abdominal surgery. However, a recent study of examining optimal tidal volume by Karalapillai, et al. added more complexity into this topic. In the study, they randomized patients to TV of 6 mL/kg versus 10 mL/kg with PEEP of 5 cmH 2 O in major noncardiothoracic, non-intracranial surgery under general anesthesia [38]. The two groups did not show any major difference in pulmonary complications. However, when they did subgroup analysis of abdominal surgery cohorts, there was a trend of less complications in the lower TV group, although statistically  [39]. Higher driving pressure was associated with the development of postoperative pulmonary complications, but neither TV nor PEEP showed any association. Importantly, an increase in the level of PEEP that resulted in an increase in driving pressure was associated with more postoperative pulmonary complications. In other words, when an increase in PEEP does not contribute to an improvement of respiratory compliance, the PEEP level can be injurious by overstretching the aerated parts of the lung.
Overall, these studies pointed out the importance of selection of optimal TV, driving pressure, plateau pressure and PEEP in the perioperative setting where patients tend to have normal lung function prior to MV as in MV.

Study design of VILI using rodents
A preclinical model is an important tool to test different ventilatory methods under controlled conditions and determine the mechanisms of VILI with the luxury of tissue sampling. A number of investigators have used different models to dissect these underlying mechanisms. Barotrauma, volutrauma, atelectrauma and biotrauma are four classic mechanisms of VILI development that have been described so far [40]. In regards to the different animal models, overall, the major difference is in the species used. Specifically, the use of large animals is effective to explore the effects of gravity on the development of VILI [6], but it is expensive and requires a large facility to accommodate. On the other hand, rodents such as mice, are much more affordable, although measuring their lung function may be more challenging than large animals [7]. However, TV, pressure and flow of rodents can be measured with commercially available equipment now. CT scan for rodents is also available for volume measurement. Here we will focus on studies describing how mice have been ventilated for mechanistic studies.
One of the major indications to use MV in mice is to provide procedural anesthesia for a short duration. For this purpose, most of the experiments have used respiratory rate (RR) of 100 to 150/min, TV of 200-700 μL (4-20 mL/kg) and inspiratory to total cycle duration ratio (Ti/Tt) of 0.2-0.4. To study VILI with a range of TVs, the majority of mouse studies have also used short-term mechanical ventilation (Table 2 and Table 3). VILI studies are divided into one-hit (Table 2) and two-hit (Table 3) models. Two-hit models have been usually done by lipopolysaccharide (LPS) instillation or cecal ligation and puncture (CLP) surgery followed by MV. The choice between one hit and two hit models should be made based on experimental paradigm. One hit model is advantageous to directly examine lung injury by MV only, but pure clinical scenario mimicking this may be restricted to perioperative MV for surgical anesthesia. Two hit model is more realistic scenario for MV use in ICU setting. LPS has been one of the major hits used for two hit models. LPS is not necessarily an infection model, so the relevance of this model should be considered in a case by case manner. In addition, mouse is more resistant to LPS than human. As described above, interspecies differences in professional phagocyte responses, particularly in the setting of the first hit, induced by infection may be important to consider when using the two hit model. CLP model has been also used as the initial hit. at least in part attributed to the difference in the signaling pathway activation. A/J mice were more vulnerable to VILI, while Balb/c were most resistant, with C57/BL6 to be in between the two strains. This intra-species difference was associated and in accordance with the difference in WNT1-TLR4 signaling pathway activation observed between the different species.
As described above, various TVs have been used for VILI studies. How should we choose TV? During tidal breathing, the change in lung volume is TV, and the initial lung volume corresponds to the functional residual capacity (FRC). Global volumetric lung strain can be estimated as TV/FRC. FRC in ARDS patients can be very small, which signifies the importance of low TV (6 mL/kg) management, whereas patients with healthy lung in the operating room setting may have high FRC, and TV at the range of 10 mL/kg may not contribute to the strain. This may be in line with the meta-analysis study of surgical patients that did not show any role in TV [39]. FRC of healthy mice breathing spontaneously was previously determined using CT scan [44]. FRC increased between 4-6 weeks-old and then remained stable after 6

Future rodent studies
Clinical studies have been often limited to examining the correlation between ventilatory settings and postoperative outcomes, without the assessment of lung parenchyma. Ventilation heterogeneity is widely considered as a marker of pulmonary dysfunction in ARDS and VILI [46]. The heterogeneous change of lung parenchyma results in a maldistribution of ventilation. More compliant regions of the lung are prone to overventilation and over-distension. Stiffer regions of the lung are prone to under-ventilation and at risk of decruitment [47]. These mechanical burdens are associated with injurious, inflammatory responses in the lung. The association between ventilation heterogeneity and VILI is well illustrated in a number of studies of supine versus prone ventilation. Compared to ventilation in a supine position, being in a prone position improved homogeneity within the lung [48]. In patients with severe ARDS, patients ventilated in prone position showed significantly lower mortality than in supine position [49].
It is intuitive that MV can affect the already heterogeneous lung, thereby further aggravating this heterogeneity. However, does MV itself contribute to this heterogeneity in the healthy lung? It is well described that induction of general anesthesia promotes a reduction in lung volume and atelectasis formation associated with a deterioration of both gas exchange and respiratory mechanics [50,51]. Does the transition from spontaneous ventilation to positive pressure ventilation contribute to this? One of the important characteristics of spontaneous ventilation is the motion of the diaphragm. Diaphragm is contracted during expiration, thus preserving distal airway patency and avoiding/reducing expiratory atelectasis formation [52]. In addition, the dorsal and more compliant part of the diaphragm moves preferentially during spontaneous breathing, which helps to improve ventilation-perfusion matching [53]. During positive pressure ventilation in the supine position, however, the diameter of the distal airway in the dorsal part of the lung, which carries more volume than the ventral part, can become significantly narrower, leading to airway collapse. Thus, positive pressure MV itself in the setting of limited active diaphragm function can contribute to the development of lung heterogeneity, which may explain the association between ventilation settings and postoperative complications in the OR setting. An adequate level of PEEP could circumvent the airway collapse on the dependent region of the lung, while minimizing overstretches the more compliant part of the lung. Parameters such as driving pressure, PEEP and TV are output in the lung as a whole, thus, understanding the impact of these parameters on different regions of the lung should be one of the important directions in VILI research.
TV, driving pressure and plateau pressure are parameters that are measured at a static state during respiratory cycle [54]. However, respiratory rate (RR) should be also an important parameter. Flow to airway before air reaches the lung alveoli requires energy and poses stress to the airway. PEEP poses stress to the lungs at the baseline. Mechanical power is a concept developed based on the hypothesis that the degree of VILI depends on the amount of energy transferred from the ventilator to the lungs [55]. Mechanical power is determined by TV, plateau pressure, PEEP, respiratory rate and air flow and is expressed as the amount of energy per minute. A study of ICU patients requiring MV showed that higher mechanical power was associated with increased ICU mortality, in-hospital mortality and 30-day mortality [56]. Another study of ICU patients also showed that higher mechanical power was associated with increased mortality [27]. These studies supported the importance of the concept of mechanical power. Respiratory rate that has been used in the studies so far ranges from 20/min to 160/min. Increased respiratory rate can increase plateau pressure, thereby increasing driving pressure [57]. Although the selection of respiratory rate is important to keep PaCO 2 in a desired range, it needs to be chosen carefully given it could affect plateau pressure. The mechanistic analysis of the relationship between mechanical power and VILI needs to be examined extensively in the future. Pressure-controlled ventilation is likely associated with less mechanical power than volume-controlled ventilation given that resistance due to flow may be much less [58], but the data so far have not indicated any superiority of one ventilation mode over the other [59]. Overall, using optimal mechanical power (TV, plateau pressure, PEEP, RR) to achieve appropriate gas exchange without or minimizing a significant strain and stress to the focal susceptible lung should be the goal of MV. It is also important to keep in mind that the duration of MV is not taken into consideration in mechanical power. Furthermore, the duration of MV is another component to be considered extensively because the study by Szabari, et al. demonstrated the contribution of longer MV to VILI.
As the importance of MV-associated inflammatory responses in VILI pathophysiology has been demonstrated to many different studies [6] ( Table 2 and Table 3), rodent experiment addressing this concept would allow us to fully study immunological responses and other molecular mechanisms contributing to VILI for future intervention.

Conclusion
In conclusion, MV is an important medical tool but still needs sophistication to reduce complications in clinical management. Preclinical models are and will continue to be important tools to supplement the void of clinical studies, but step-by-step approaches and well-defined and different combination of parameters will be necessary for optimal translational potential in order to obtain the correct answers for our clinical questions.

Figure 1:
The relationship between pressure and time, and between pressure and volume.    Rodent two-hit VILI model. Ding, et al. [73] Transl Perioper Pain Med. Author manuscript; available in PMC 2022 January 05.